i) Field of the Invention
The present invention relates to a primer for detecting methicillin-resistant and/or toxic shock syndrome toxin 1 producing Staphylococcus spp., a method for detecting these bacteria using the primer, and a kit for detecting them.
ii) Description of the Background Art
Staphylococcus spp. including Staphylococcus aureus as a typical bacterium, which have strong pathogenicity, are well known as causative bacteria to various infections. Since these Staphylococcus spp. are generally sensitive to .beta.-lactam series drugs, the infections can be prevented and treated with these drugs. However, a methicillin-resistant Staphylococcus sp. (abbreviated as MRS hereinafter) is so extensively resistant to the .beta.-lactam series drugs that it is difficult to treat infections caused by these methicillin-resistant bacteria. This provides serious problems that the methicillin-resistant bacteria cause opportunistic infections, postoperative infections and the like in the clinical practice.
Since the .beta.-lactam series drugs have high safety and wide antibacterial spectra, some of the .beta.-lactam series drugs are widely used as primary choices of drugs against various infections. The mechanism of these drugs is that binding of the .beta.-lactam series drugs to cell wall-synthesizing enzymes (penicillin-binding proteins: PBPs) which are essential for growth of bacteria results in inhibition of the growth of the bacteria. However, the methicillin-resistant bacteria have changed to produce an additional low-affinity penicillin-binding protein, PBP-2'[Hayes, at al., FEMS Microbial. Lett., 30, 119-122(1981)], which is active at .beta.-lactam concentrations that saturate normal complement of PBPs, so that these bacteria are resistant to .beta.-lactam series drugs.
It is very important to determine whether infectious bacteria are resistant to methicillin in order to set up a therapeutic procedure against the bacteria, and recently susceptibility tests by culturing bacteria have been widely used as methods for detecting MRS. The culturing method, however, requires 2 days to determine the susceptibility. Since some drugs are needed to be administered to patients prior to the determination, incorrect therapy may lead patients to die because effective drugs for MRS are limited. Thus, methods for rapidly detecting MRS should be developed without delay. Moreover, Expression of methicillin resistance in susceptibility tests is subjected to environmental conditions such as temperature [Canawati, H. N. et al. Antimicrob. Agents Chemother. 21, 173-175(1982)], PH [Sabth, L. D. et al. Antimicrob. Agents Chemother, 2, 350-355)] and salt concentration [Chambers, H. F. et al. Antimicrob. Agents Chemother. 31, 1982-1988(1987)]. Conditional expression of PBP-2' may cause ambiguities in susceptibility tests [Barry, A. L. et al. J Clin. Microbiol. 25, 1897-1901(1987)].
Under the present circumstances, researchers have tried to detect MRS by genetic techniques. One of them is an approach that detects the gene for PBP-2' (mecA gene) characterizing MRS [Tomasz et al., Antimicrob. Agents Chemother., 35; 632-639 (1991)]. However, it is difficult to apply the method, which uses the dot hybridization with isotope-labeled DNA probes, in clinical institutes such as hospitals in view of handling. On the other hand, there is another approach [Higashiyama et al., 65th Nippon Kansenshogakkai Kouen Shouroku, p. 13 (1991)] using a polymerase chain reaction [PCR; Mullis et al., Science, 230: 1350-1354 (1985)], which is a highly sensitive and relative rapid method for detecting various genes, though complicated handling and difficult treatment of multiple samples makes the approach unpractical.
Among MRS, strains producing toxic shock syndrome toxin-1 (abbreviated as TSST-1 hereinafter) have been isolated at high frequencies, TSST-1 producing Staphylococcus spp. is abbreviated as TPS hereinafter, and it is said that the mortality of the patients carrying these strains is high. There is a general method for detecting the toxin-producing Staphylococcus spp., which directly detects the toxic protein by the immunological method [See et al., J. Clin. Microbiol. 27:2050-2053 (1989)], and whose sensitivity depends on the amount of toxic proteins produced, being insufficiently used at clinical institutes. The method, which is based on culturing, is also insufficient in view of rapidness. Subsequently, TSST-1 gene was isolated [Schliever et al., J. Biol. Chem., 261: 15783-15786 (1986)]. Although the detection of Staphylococcus aureus producing TSST-1 is tried by detecting its gene, this detection method is not practical because of the same reasons as those of the above-mentioned method for detecting MRS.